RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved]
Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challe...
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| Format: | Article |
| Language: | English |
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Wellcome
2017-07-01
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| Series: | Wellcome Open Research |
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| Online Access: | https://wellcomeopenresearch.org/articles/2-34/v2 |
| _version_ | 1819262133673656320 |
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| author | Clémentine Delan-Forino Claudia Schneider David Tollervey |
| author_facet | Clémentine Delan-Forino Claudia Schneider David Tollervey |
| author_sort | Clémentine Delan-Forino |
| collection | DOAJ |
| description | Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads. |
| first_indexed | 2024-12-23T19:52:51Z |
| format | Article |
| id | doaj.art-04017f74e712423482b8ccc7afd1bee4 |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-12-23T19:52:51Z |
| publishDate | 2017-07-01 |
| publisher | Wellcome |
| record_format | Article |
| series | Wellcome Open Research |
| spelling | doaj.art-04017f74e712423482b8ccc7afd1bee42022-12-21T17:33:19ZengWellcomeWellcome Open Research2398-502X2017-07-01210.12688/wellcomeopenres.10724.212970RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved]Clémentine Delan-Forino0Claudia Schneider1David Tollervey2Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3BF, UKInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UKWellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3BF, UKBackground: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC) was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly disfavored for the generation of short reads.https://wellcomeopenresearch.org/articles/2-34/v2BioinformaticsCell Signaling & Trafficking StructuresGenomics |
| spellingShingle | Clémentine Delan-Forino Claudia Schneider David Tollervey RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved] Wellcome Open Research Bioinformatics Cell Signaling & Trafficking Structures Genomics |
| title | RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved] |
| title_full | RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved] |
| title_fullStr | RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved] |
| title_full_unstemmed | RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved] |
| title_short | RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved] |
| title_sort | rna substrate length as an indicator of exosome interactions in vivo version 2 referees 3 approved |
| topic | Bioinformatics Cell Signaling & Trafficking Structures Genomics |
| url | https://wellcomeopenresearch.org/articles/2-34/v2 |
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